JPH05501058A - Antisense oligonucleotide inhibition of papillomavirus genus - Google Patents

Abstract

Oligonucleotides and oligonucleotide analogs are provided which are capable of antisense interaction with messenger RNA of papillomavirus. Such oligonucleotides or oligonucleotide analogs can be used for diagnostics and therapeutics as well as for research purposes. In accordance with preferred embodiments of this invention, oligonucleotide or oligonucleotide analog is provided which is hybridizable with a messenger RNA from a papillomavirus. The oligonucleotide or oligonucleotide analog is able to inhibit the function of the RNA, and accordingly is useful for therapy for infections by such papillomavirus. In accordance with a preferred embodiment, portions of the papillomavirus are targeted for antisense attack. Thus oligonucleotides are preferably provided which hybridize with the E2, E1, E7, or E6-7 messenger RNAs.

Description

[Detailed description of the invention] Antisense oligonucleotide inhibition of papillomaviruses Field of the invention The present invention relates to the inhibition of papillomaviruses and the The present invention relates to the diagnosis and treatment of infections in animals in which Papillomavirus spp. Detection and quantification of milk BXm viruses in Ti: samples suspected of containing do.

Furthermore, the present invention provides methods for inhibiting the function of metsusenger RNA derived from the papillomavirus genus. for oligonucleotides and oligonucleotide analogs that Ru. Such interference may be used as a means of diagnosing and treating papillomavirus infections. I can be there. It is suitable for both research and diagnostic use as well as for test reagents. It can also form the main component for a kit.

Background of the invention The genus Papillomavirus (PA) is widely distributed in nature and generally It is associated with benign epithelial and fibroepithelial lesions, commonly referred to as epithelial lesions. They are humans, It has been detected in a variety of higher mammals including cows, rabbits, deer and several bird species. and isolated. Although these viruses are generally associated with benign lesions, , certain types of viruses have been linked to lesions that can progress to cancer.

These viruses may play an etiological role in the development of certain human cancers. The close relationship between transcriptionally active human papillomavirus (HPV) deoxyribo The results of numerous studies have shown that nucleic acids are present at high rates in certain cancerous lesions.

Zur Hausen, H. and Schneider E. (Schneider, A, ＞1987.The Pa ovavirida e.

Volume 2, N.P. Salzman and B.A. Edited by P. M. Howley, pp. 254-264, Plenum P. Plenum Press, New York.

In humans, human papillomavirus causes common warts on the hands and feet, as well as laryngeal warts. There are currently more than 57 types of HPV that cause various diseases including genital warts and genital warts. It was confirmed in Each HPV has a preferred anatomical infection site, and each virus , generally may be associated with specific pathologies. Venereal warts and genital warts Genital warts, also called genital warts, are one of the most serious manifestations of Pv infection. disease management center The mode of sexual transmission of genital warts is well established, as reported by the Internet. The incidence of genital warts is increasing. The importance of genital warts is that HPV DNA is What can be found in all grades of epithelial neoplasia (CIN I to III) and recent evidence that certain types of HPV species can be found in cervical cancers. highlighted by the findings of Therefore, genital warts containing certain HPV species Certain women are currently considered to be at high risk for developing cervical cancer. Genital warts Current treatments for the disease are inadequate.

There is great hope in providing compositions of substances capable of interfering with Papillomavirus spp. There are, but so far unexplored, similar findings regarding the papillomavirus genus of animals. It is desirable to achieve therapeutic and diagnostic methods for this disease. In addition, papilloma will There is a need for improved kits and test reagents for use in research on the genus.

Purpose of invention The object of the present invention is to hybridize with Metsenger RNA of the genus Papillomavirus. oligonucleotides that can inhibit the function of metsenger RNA. and oligonucleotide analogs.

Another goal is antisense interaction with viral metsenger RNA. oligonucleotides that can modulate the functional expression of papillomavirus DNA by An object of the present invention is to provide cleotides and analogs.

Another object of the invention is a diagnostic method and method for papillomaviruses in animals. and to provide treatment.

Detecting the presence or absence of Papillomaviruses in a sample suspected of containing them Methods, materials, and kits for the development of the methods, materials, and kits are another object of the present invention.

Novel oligonucleotides and oligonucleotide analogues of the invention It is a purpose.

These and other objectives will be realized upon consideration of this specification and the appended claims. will be clear to those skilled in the art.

Brief description of the drawing Figure 1 is a schematic map of the genetic regime of some Pv genomes.

FIG. 2 is a partial genetic map of BPV-1, a bovine papillomavirus.

The genome shows open reading frame 0RFs, and Metsenger RNA is transcribed from that genome. has been done.

Figure 3 shows the BPV-I E2 transcript showing nucleotides 2443 to 4203. This is the nucleotide sequence of the activator gene mRNA.

Figure 4 shows the BPV-domain with nucleotides 2443 to 3080t. I This is the nucleotide sequence of E2 transactivator gene mRNA.

Figure 5 shows the initial messenger domain with nucleotides 89 to 304. The nucleotide sequence of the 5' common untranslated region of BPV-1, which encodes be.

Figure 6 shows the nucleic acid of antisense oligonucleotides produced by the techniques of the present invention. nucleotide sequence and the relative position of the oligonucleotide on E2 mRNA . The oligonucleotide identification, sequence and functional role are described.

FIG. 7 shows the E of antisense oligonucleotides produced by the techniques of the present invention. This is a graph showing the effect on 2 expression. *Targeting the CAP region of mRNA Translation of oligonucleotide (11751) and E2 transactivator Codon (11753) is the E21-lance activator at micromolar concentrations. It can be seen that this reduces the

FIG. 8 shows the use of antisense oligonucleotides produced by the techniques of the present invention. It is a graph showing quantity response.

These dose-response curves show that E2 transactivation in the region containing the translation initiation codon antisense oligonucleotides that are complementary to cation messenger RNA The 50% inhibitory concentration (IC5o) of 11753 is 50-100 nanomolar; oligonucleotide (11751) targeting the CAP region of one message. IC5o is in the range of 500 nanomoles.

Figure 9 shows the transactivation of E2 mRNA produced by the technique of the present invention. Antisense oligonucleotides targeting the mother region and the mother region This is the nucleotide sequence of Otide.

Figure 10 shows selected samples targeting the transactivator region of E2 mRNA. 1 is a graph showing the effect of oligonucleotides produced by the technique of the present invention. The 15-20 mer antisense oligonucleotide was has been shown to inhibit tivation.

Figure 11 shows the selected is a graph showing the effect of oligonucleotides produced by the technique of the present invention. The 15-20 mer antisense oligonucleotide It has been shown to inhibit the

Figure 12 shows the biological effects of BPV-1 on the reduction of E2)-lance activator in the field. This is a photograph showing the results of antisense origin produced by the technique of the present invention. The oligonucleotide inhibits or reduces BPV-1 transformation of C1274 [B cells. was tested for ability to

Figure 13 shows selected samples targeting the transactivator region of E2 mRNA. is a graph showing the effect of oligonucleotides produced by the technique of the present invention. Inhibition of BPV-1 focus formation by antisense oligonucleotides These dose-response curves for 11751 and 11753 show that The IC of 11753 is in the range of 10 nanomolar, while the IC5o of 11751 is in the range of 10 nanomolar. It is shown to be in the range of 0 nanomole.

Figure 14 shows the effects of the present technique on the ability of BPV-1 to replicate its genome. 2 is a graph showing the effects of antisense oligonucleotides produced by Wi Cells transformed by Virus were treated with 11751 and 11753 and The DNA of the virus was quantified.

Figure 15 shows cells treated with metabolically WA-identified oligonucleotides or untreated. The results of an immunoprecipitation assay in which a control sample was immunoprecipitated using a monoclonal antibody are shown.

Figure 16 shows the nucleotide sequence of HPV-11 in the region of the translation start codon of E2. be.

Summary of the invention Preferred implementation of the invention! 9′m, Metsenger from the genus Papillomavirus Oligonucleotides and oligonucleotides capable of hybridizing with RNA provide analogs. This relationship is generally referred to as "antisense". e). Oligonucleotides and oligonucleotide analogs are RN A's function is translation into protein, translocation to the cytoplasm, or its biological function. Any other activity required overall can be inhibited. Metsenger RNA Inability to perform all or part of its functions, the papillomavirus genome will not be properly expressed, i.e. will not multiply and the correct progeny will be valid. are not formed in numbers, and such infections caused by animals infected with Papillomavirus spp. The deleterious effects of the offspring are modulated.

Here, we show that the genes expressed by several metsenger RNAs of the papillomavirus genus It turns out that it is preferable to target that part of the genome for antisense attacks. won. Here, mRNA of E2, El, El and E6-7 of papillomavirus genus It has been found that A0 is particularly suitable for this study, e.g., oligonucleotides or the desired method for hybridization with oligonucleotide analogues. Senger RNA is E2, El, El or E6-7 Metsenger RNA According to an even more preferred embodiment, bovine papillomavirus -1 from nucleotides 2443 to 3080 or 89 to 304 The E2)-transactivator region of papillomaviruses, such as those exemplified by or is designed to be complementary to the RNA portion transcribed from the 5′ common untranslated region. Oligonucleotides and oligonucleotides containing nucleotide base sequences provide analogs.

Most preferred implementation! mRNA for E2 transactivator by g-like A. Oligonucleotides targeting the CAP region and translation codons are provided.

These oligonucleotides span nucleotides 2443 to 418 of BPV-1. Designed to hybridize with E2 mRNA encoded by up to 0 .

Here, the method for modulating the expression of Papillomavirus genus is the method for modulating the expression of Papillomavirus genus Metsenger RNA derived from the Papillomavirus genus Oligonucleotide or oligonucleotides capable of hybridizing with NA contacting the oligonucleotide or oligonucleotide with the analog; When hybridized thereto, the methsenger RNA It was discovered that it inhibits function. Papillomavirus genus E2, El, El or Oligonucleotides designed to hybridize with E6-7 mRNA It is preferred to use otides or oligonucleotide analogs.

Furthermore, herein a method for modulating the effects of papillomavirus infection in animals is described. of animals were hybridized with metsenger RNA from the genus Papillomavirus. contact with an oligonucleotide or oligonucleotide analog that , thereby, when hybridized thereto, said Metsenger RN It was discovered that it inhibits the function of A. E2, El, Elma of the papillomavirus genus or oligonucleotide or oligonucleotide that can hybridize with E6-7 mRNA. Ligonucleotide analogs are preferred.

Papillomas using such oligonucleotides or oligonucleotide analogs Diagnostic methods for detecting the presence or absence of virus spp. Kits and test reagents for this type of diagnostic activity by hybridization As with drugs, they are within the scope of this invention.

Detailed description of the invention Genital warts are the most frequently diagnosed viral sexually transmitted disease. clinically They are divided into two main groups, namely condylomata acuminata and condylomata acuminata. It can be classified as a wart. Condylomata are known to contain virus particles. Molecular studies have shown that over 90% of these lesions are HPV-6 or It was demonstrated that it contained HPV-11 DNA. Gls″sm ann, L.), Wolnik, L., Ikenberg, E. Ikenberg, H., Koldovsky Knee, ov. sky, U,), Schnurch.

H, G.) and Zur Hausen, H.

Proc, Nat 1. Acad, Sci, USA, 80,560-563 ( (1983), genital warts generally occur on the penis, vulva, or perianal area. arise. They may recede on their own or persist for years, leading to erosive cancer. progression occurs only infrequently. Unlike other genital warts, those that occur on the cervix has a flattened rather than cuspate morphology and is usually detected clinically on a Papanicolaou smear. Served. The genus papillomavirus, which is the cause of cervical dysplasia, was discovered in the 1970s. In the terminal stage, Baba Nikolausmi with cytological changes and dysplasia due to HPV infection. This was suggested by research by cytologists who demonstrated a relationship in A. Fl! In the first study , many of the dysplastic cells in these lesions contain viral particles and viral capsid antibodies. It was shown that the origin exists. This relationship has been shown in previous clinical studies to CIN (also called CIN or cervical epicutaneous neoplasm) is a precursor to cancer in the field. In turn, it has been recognized that it is a precursor to invasive squamous cell carcinoma of the neck. This is important because it confirms what has been said. HPV types 16 and 18 cause cervical cancer Directly cloned from . Deurst, M, Gisma Boshart, M., Gizman L., Ikenbar G H, Kleinhelnz, A, Schuerle Scheurfen, W. and Zur Hausen H. EMBOJ, Dan, 1151-1157 (1984). continues to be used as a sation probe to detect over 70% of human cervical cancers and We show that the induced cell lines are positive for the presence of both these HPV types. did. The other 20% include additional HPV types, such as HPV-31, HPV-3 3 and HPV-35.

Data collected from the U.S. Treatment Index showed that in 1984, the first diagnosis of genital warts 224,900 cases and 156,720 cases of first diagnosis of genital herpes. It was. The average incidence of genital warts from 1950 to 1978 was ioo, ooo As demonstrated by epidemiological studies reaching 106.5 per capita , has steadily increased from 1970 to 1980. 25 to 55 years old The prevalence of cervical HPV infection among women aged 22 years was 0.8%; ．． It was 7%. Recent studies in cytologically normal women have shown that latent infection The incidence was demonstrated to be 11%. Therefore, higher incidence and There appears to be an incubation period for the disease that suggests morbidity.

Active genital warts can be identified in about 2.5% of pregnant American women, and It is therefore associated with 60,000 to 90,000 pregnancies per year. HP ■ PV is more prevalent in pregnant women than three times. Every year, severe cases of laryngeal papillae occur. With an estimated 1,500 new cases, the risk of transmission from mother to newborn is 8. 0-1: Indicates 200.

Laryngeal papillomas are benign epithelial tumors of the larynx. There are two types of PV, namely HPV- 6 and RPV-11 are most commonly associated with laryngeal papillomas. Clinically, Laryngeal papillomas are divided into two groups: juvenile onset and adult onset. classified. In the case of minors, newborns were infected with genital P■! through the parent's birth canal It is thought that the infection can occur once the infection has passed. The disease is usually detected at age 2 and is a benign papilloma. grows slowly but steadily and eventually obstructs the airway without surgical intervention. It is characterized by These children typically have multiple recurrences of the papilloma. will also undergo surgery. Eventually, the patient succumbed to complications from repeated surgeries. Ru.

Currently, there is no cure for laryngeal papillomatosis that develops in minors, and natural Regressions are rare. Laryngeal papilloma that develops in adults is not aggressive and has a spontaneous remission course. I often make mistakes.

The most common disease associated with papillomavirus infection is benign skin warts.

In general, common warts include HPVI type, type 2, type 3, type 4 or type 10. These warts typically occur as plantar warts that occur on the soles of the feet or on the hands. general Skin warts are most commonly found in children and young adults, and become more common in later life. The incidence of warts probably decreases due to immunological and physiological changes. Plantar warts can often be debilitating and require surgical removal. Currently, there is no treatment for plantar warts, which often recur after surgery. There is no reliable treatment for common warts, which are ugly but rarely debilitating. Very rarely, therefore, it is generally not treated surgically.

Epidermal dystrophy (EV) is a rare genetically transmitted disease that causes small red spots. EV is characterized by scattered flat warts that appear as dark spots. Approximately one-third of EV patients develop skin lesions in multiple areas over time. develop squamous cell carcinoma (SCC). Generally, SCC is associated with sun exposure of the skin Occurs in parts. Only EV-related types of PV were found in SCC, HPV-5 and HPV-8. Genetic predisposition, immunological changes and UV irradiation and HP Both V may contribute to the development of SCC in these patients.

The P genome is a covalently closed double-stranded structure with approximately 8,000 base pairs. It consists of a circular DNA molecule. Complete nucleotides of numerous animal and human PVs The sequence and genetic system have been determined for bovine papillomavirus type 1 (BPV-11, etc.) Ta. Chen, E, Y, Hawso P.M. owley, P. M.), Levinson A.D.

A, D.) and Seeburg, P. H., N. ature, 299.529-534 (1982), with some PV schematic maps. As shown in Figure 1, viral transcription is unidirectional, i.e., the entire viral mRNA Viral DNA with the same parts! Transcribed from If. Engel El W (Enge l, L, W,), Hei Iman, C.

A,) Oyohiha’y’)--h”--M, J, Viro 1..47. 516-528 (1983); Beilman C.A., Engel L., Lo. Lowy, D.R. and Howso P.M., brother 1-1119.22-34 (1982), The genetic code chain is an open reading frame ( 1 individual 0RFs, including 10 types called ORFs), can infect productivity. Pv genomes and their expression patterns in non-productive versus non-productive cells. Figure 2 is classified as ``early J'' or ``late'' based on their location. , shows the relationship of some 0RFs of bovine papillomavirus-1.

Ebiso in the cells transformed with the odontodont 4I follicle and its genome transformed. Because of its ability to persist as a tumor, BPV-1 has become a model papilloma in in vitro studies. It served as a virus genus. As a result, BPV-1 is a total papilla-spreading virus. This is the one that shows the most characteristics. The BPV-1 genome is 7946 base pairs long. were cloned and sequenced. Choi 2 et al., 1982. Above, B DNA sequence analysis of PV-1 reveals eight early (E) and two late (L)! The designation of 0RFs as early or late is permissive. Their expression patterns in nonproductively infected transformed cells versus normally infected cells Based on turns. Beilmann et al., 1982. Above; Baker C.C. (B aker, C, C,) and Howto B.M., EMBOJ, 6゜102 7-1035 (1987).

All 0RFs have the same DNA! The mR contained on a and currently characterized It was shown that the entire NA is transcribed from the genetic code strand. Amtman Ei It has been found that some ORFs have multiple functions. E5 and E6 are ( Lusky, M.) and Botchan, M.

Code the modulation cipher of the DNZ copy. Robert J.M. S, J. M.) and Weintraub, H. Ce11. Life 6,741-752 (1986).

The full-length E20RF is a protein that transactivates viral transcription. [Spalholz, B, A, ), Yang Y. C. and Howto B. M., Ce, 42, 183-1 ．． 91 (1985)], and 3'E2 0RF is a transducer of viral transcription. Code a suppressor. Lambert, P.

F.), Sparholz B.A. and HOWTO B.DATABREW.

Ce1l, Dandan, 69-78 (1987), E3, E4 and E of BPV-1 The function for 8 has not yet been determined.

Ll [Cowsert, L.M., Pirajński W B (Pi 1acinski, W, P,) and Jis’/Son A・B (Jenson, A, B,), vtroshi2jLL・　16th・6 13. Oligonucleotides and oligonucleotides targeting or in part This is complicated by the existence of similar price patterns. So far, 18 types Different classes of mRNAs were determined by various methods. Amtman Eo and Sour G., J., Virol, Dan, 59-66 (1982); Burnett, S., Moreno, Robets, J. – C.A., Engel L., Rollei D.R. and Howto B. -M, Virolo, 1, 9.22-34 (1982); - C and Howto B.M., EMBOJ,, Dan, 1027-1035 (1987), Stenlund, A., Zabielski ・Jay (Zabielski, J,), AhoIa (H,) , Moreno-Lopez, J. and Petterman-N., J., Mo1. Biol.

1dan 2,541-554 (1985); and Y. Yang, Okayama. H and Howto BM, Proc, Natl Acad, Sc1゜ and The 5' end of the other mRNA5 contains the common region between nt890.2443 and Capsids are non-enveloped, non-glycosylated, LL and L2 The capsid is made up of 28M virus-encoded proteins called . Ll is the major capsid protein and is one of the proteins present in the lion. 80% and its molecular weight is 50-60 kD. L2 is a small amount of capsidota It is a protein and has a theoretical molecular weight of 51kD, but it has been found to migrate to 76kD. won. Pv cannot be produced in vitro and even in productive lesions. Since very few mature pillions are found in PV, detailed information on the serology of PV is lacking. It was impossible to conduct detailed research.

The life cycle of papillomaviruses is complex and currently poorly understood. Currently, there are no in vitro systems that allow the production of mature P. pillions. As a result of the inability to characterize the life cycle of the papillomavirus genus, It had not been developed. PV has a limited host range and rarely crosses species barriers. Furthermore, PV infection only discriminates between mucosal or stratum corneum epithelium. P ■ Regulation of gene expression is thought to be first linked to the discrimination program of host epithelial cells. available. The currently preferred model for the P ■ life cycle is as follows, all of which are That is, infectious 17 particles penetrate the outer epithelial layer through trauma and penetrate the basal lining of the host tissue. infect the cells. Viruses are maintained as relatively low copy extrachromosomal elements.

When epithelial cells begin to undergo differentiation, the PV genome is replicated to a high copy number and the cell terminates. When the late stages begin to undergo discrimination, late genes are expressed and the viral genome changes. encapsulated.

The papillomavirus genus was discovered to be an ideal target for antisense therapy. . First, papillomavirus lesions are external and antisense oligonucleotides Allows for localized testing of titanium and eliminates many problems such as rapid clearance. rIIJ (X oligonucleotide) and systemic administration of synthetic oligonucleotides. A clinically active group II concentration of nucleotides is obtained. Second, the viral genome It is maintained as a separate genetic element in the infected cells. This is recommended to treat symptoms. As proposed, attacking the replication function of the virus could lead to a potential cure. It opens the door to

E20RP of the papillomavirus genome is particularly useful for antisense oligonucleotides. It was discovered that there was enough left in Chido's design. E2 is BPV-1 and HP It appears to be the major transactivator of viral transcription in both the V and V systems. Do you get it. Mutation of E20RF impairs transformation and extrachromosomal DNA replication. It has pleiotropic effects. DiMaio, D, + J .

Virol, Dane, 475-480 (1986); and Settleman, J., EMBOJ, 7゜11 97-1204 (1988); Grob. Groff, D.

E.) and Lancaster W.D.

D, ), Vi Nitachijodan Ei, Vol. 50, 221-230 (1986); Rapson ・M Nis (Rabson, M, S,), Yee, C, Yang - Rai C and Howso B.M., J. Virol,, average, 626-6. 34 (1986); and Sarver, N., Rapson. - M. Nis, Yang Lai C., Byrne, J.

C, > and Howso B.M., J. Viro 1. ．． 52.37'7- 388 (1984), subsequently E20RF activates transcriptional transactivators. I don't know how to code, Svarholz B.A., Yang Lai C. and Howso BM, Ce11. Dan, 183-191 (1985), internal The truncated translated portion of E2 resulting from initiation of translation at the AUG of It turned out to be a trans-repressor. Lambert B.F., Subaru Holtz B.A. and Howso B.M., Ce1l, Σdan, 69-78. (1987>, the carboxy-terminal 100 amino acids that are the DNA-binding domain) [McBride, A.A., Schiegel, A. Schlege7.R, and Howso BM, EMBOJ.

E, 533-539 (1988>) and the DNA recognition sequence ACCN60 GT [Androphy, E, J,), Q-Ra I.D.R. and Schiller, J. , T.), Nature, 32nd, 70-73 (1987) and Moskaluk. ・Moskaluk, C. and Bastia Dee a, D, ), Proc, Natl, Acad, Scf, USA, Dan 4.1215 ~1218 (1987>) was determined.The E2 transcriptional regulatory circuit was These are general characteristics of each genus. E2 transregulation has been This was demonstrated in each of the other papillomavirus genera tested. Gius Di - (Gi, us.

D.), Grossman S., Bedell M.A. Bede 11. M, A, ) and Lai, m1ns .

L, A heat, J, Viro 1. ．． 62.665-672 (19881; Hiro Hirochika, H, > Broker T.R. roker, T, R,) and Chata L.T.<Chow, L, T,), J, Viro 1. ．． 61.2599-2606 (1987); Chin M ・Tea (Chin, M, T,), Hirochtk a, R,), Hirochika H, Broker T.R. and Chata E. Le T., J. Virol, 62.2994-3002 (1988); Ellas Dubliny Sea (Phe lps, w, C1) and Howso Bi -xmu, positive.

Virol, 61.1630-1638 (1987); ・Thierry, F. and Yaniv, M. EM Dan OJ, 623391-3397 (1987).

In the present invention, the obligatory viruses shared by animal and human papillomaviruses, respectively, Differentiation of viral transcriptional elements indicates that E2 is useful for testing, diagnosis, and treatment of papillomavirus spp. This makes it the most important target for antisense approaches. It was decided that

The present invention further provides that the E1 locus is a location for attack by papillomaviruses. was determined to be promising. BPV-1 transformation of M. odontocephalid fibroblasts Initial sudden change assays identified El as a candidate regulator of viral DNA replication. It was decided as a tar. 3'E1 0RF is required for viral genome replication and maintenance. Code the necessary factors. Server F, Loveman M Nis, Yang Lai. C., Bahn J. C. and Howso B. M., J. Virol.

Σ2, 377-388 (1984) Nilasky, M. and Bochan, M. Earl, J. Virol, 5U, 955-965 (1985); and Russ. K. M. and Bochan M. R., J. Virol, 60.729~ 742 (1986), inhibition of the expression of E1 transcripts inhibits BPV-1 (and potentially ) (PV) that inhibits its ability to replicate its DNA in uninfected cells. It is considered that

The inventors further believe that the E7 region is useful for the treatment, diagnosis and testing of papillomavirus spp. Deciding that this would probably provide another entry point for BPV-1. In this study, El was found to be involved in the regulation of viral DNA replication.

Berg, L. J., Singh.

K.) and Bochanm, Mo1. eel 1. Biol,, 6.859~ 869 (1986), in HPV-16, El is responsible for transformation and immortalization. It has been proven that it is related to Although it is not clear at present, the E of HPVs However, if E7 specificity is involved in the replication of viral DNA, Antisense oligonucleotides and analogs inhibit BPV-1 viral DNA replication. It is thought that it inhibits

The E6-E7 region of HPV was found to be a transforming region. life of virus The exact role of this region in the active ring is currently unknown, however , this area is important in the biology of antisense of virus-induced lesions. oligonucleotides targeting this region are also likely to be It has been determined that the following examples are similarly useful for the purposes of the present invention.

Recent studies have shown that the 5' region of mRNA5, preferably the cap region and start co- Antisense oligonucleotides directed against Don block gene expression. It has been suggested that this is the most effective way to harm A transcript of the papillomavirus genus One feature is that many mRNAs have a common 5' untranslated region and cap. That's true. Therefore, antisense oligonucleotides directed against this region It was determined that leotide has the potential to incapacitate a large number of mRNAs. A large number of cormorants Inhibition of viral genes is likely to occur in additional ways and from infected cells. probably act synergistically to a minimal extent in the radiation of the viral genome of .

Antisense attacks by implementing certain preferred embodiments of the invention The OR, Fs of the papillomavirus genome yielding mRNA5, a preferred target for It will be understood that the mRNAs encode portions of other mRNAs as well.

The aforementioned 0RFs are summarized in Table 1.

deep red Papillary virus genus open reading frames and functional ORFs attributed to them! I (1 genus function E (BPV-1> (3' part) replication (BPV~1) E2 (full length) transcription trans activation (BPV-1, HPV-6, HPV-16> (3' part) Transcription inhibition (BPV-1) E4 Cytoplasmic phosphoprotein in warts ( HPV-1) E5 transformation, stimulation of DNA synthesis (BPV-1) E6 transformation (BPV-1) El　Plasmid copy number control (BPV-1>Ll　Major capsid protein L2 Small amount of chaacid protein The present invention provides antisense inhibition of the function of metsenger RNA of the papillomavirus genus. Oligonucleotides and oligonucleotide analogs for use in.

In the context of the present invention, the term "oligonucleotide" refers to the native phosphonucleotide Naturally occurring bases and cyclofuranisyl groups linked by ester bonds means a polynucleotide produced from This term refers to naturally occurring SI or synthesized from naturally occurring subunits or their close homologs. Effectively means mature.

When the term "roligonucleotide analogue" is used in connection with the present invention, oligonucleotide Functions similarly to nucleotides, but has a non-naturally occurring moiety, and is closely It means parts that are not homologous. Therefore, oligonucleotide analogs The minute or in-cylinder coupling may be modified; these examples are examples of those used in the art. phosphorothioates and other sulfur-containing species known to be some According to a preferred embodiment, at least some of the oligonucleotides are The ester bond is located in the RNA whose activity is to be modulated! Penetrates the area of the cell that is substituted with a structure that functions to increase the composition's ability to Placement like this The conversion can be performed using phosphorothioate linkages, methylphosphothioate linkages or short chain alkyl linkages. According to a preferred embodiment, it preferably comprises a cycloalkyl or cycloalkyl structure, Mention may be made of species containing radical forms other than those normally found in nature, e.g. Purines and pyridines can be so used. Similarly, nucleoti The modification of the cyclofuranose moiety of the desubunit adheres to the essential idea of the present invention. may occur as long as the

Such analogs can be made from natural oligonucleotides (or from natural derivatives). According to the present invention, as long as the RNA functions to inhibit the function of the RNA, ing.

The oligonucleotides and oligonucleotide analogs of the invention are useful for papillomatous viruses. It is designed to hybridize with Metsenger RNA of the genus. This way If hybridization is achieved, the normal interfere with its functionality, rendering it useless against viruses. Obstructed message The functions of index RNA include all biological functions, such as protein interrogation. translocation of the RNA to the position, the actual translation of the protein from the RNA, and the There are probably quite independent catalytic activities that can be involved. This is the RNA function. The overall effect of interfering with the virus is to cause the papillomavirus to lose its RNA advantage and its The overall effect is to experience interference with the expression of the viral genome.

Such interference is generally fatal.

According to the present invention, metusene has been determined to have increased metabolic importance for viruses. Oligonucleotides and oligonucleotides designed to interfere with jar RNA Preferably, leotide analogs are provided, such as El, E, as described in T#. 2. Elmana E6-7 papillomavirus mRNA5 is a preferred target.

Furthermore, 2443 to 3080 or 89 of the bovine papillomavirus-I genome encoded by nucleotides substantially equivalent to the nucleotides in et al. Interfering with the RNAs that led to them leads to the large number of RNAs that lead to essential proteins. These are particularly vulnerable to attacks if they are considered to be coded. The preferred papillomavirus genus is bovine papillomavirus, which is thought to represent a Although it has a somewhat different structure from Virus-1, essentially similar parts are commonly used. It is understood that this can be determined in any number of ways.

Figures 3, 4 and 5 show these parts of the bovine papillomavirus-1 genome. Both have particular utility in interfering with the action of such papillomaviruses. It is considered to have. References targeted by 82 mRNA of bovine papillomavirus-1 Typical oligonucleotides are shown in Table 2.

Roaring Dan BPV-1, E2 antisense oligonucleotide oo5 (Lcoos, x+n ) AAATGCGTCCAGCACC to CCATGGTGCAGT l-ra Starting point of Nsligretsa 006 (LCOO6, ABl) AGCACCGGCCATGGT Transli Gresser 014 (LC014, input ax) GGACAGTCACCCCTT 5’ Code Nagiguchioro1s (Lco1!i, AJI4) TGTACAAAT TGCTGTM; ACAGTGT entered CCAC? Intermediate code area; 016 (LC O16, AEl) αgo GTAGACA to TGTA intermediate code region 017 (LC O17, ABl) GTGCGAGCGAGGACCGTCCCCGTACCCM CC3': 7-do W7I area 01B (LCOLB, ABl) G (:ACCGTC CC(:TACC3' Code nail 0 or 019 (I, CO19, ABl)'!Tr AACAGGTGGAATCCATCATrGGT to GTG 5': 7-9 R area 020 (LCO20, ABl) GG Ayu TccxTcATTcc 5’ code A typical oligonucleotide targeting the translation initiation codon of HPV-11 Antisense oligonucleotides targeting the HPV-11 translation start codon Chido [H#! t8+1 number 5'------1--------3' identification arrangement Therefore, the 200 types of oligonucleotides shown above (or their analogs) ), or one of ordinary skill in the art, as mentioned above, From the knowledge of the preferred regions of E20R and F, it is possible to manufacture It is preferred to use any of the similar oligonucleotides (or analogs) that can From the knowledge of the sequence of each of these regions, we can create a similar table for papillomas. Other preferred 0RFt' of the virus genus! Regarding target, El, El and E6-7 can be created.

The oligonucleotide or analog is exactly as described in the table above - or necessitated by an unoriginal interpretation of the map of the papillomavirus genome. Rather, the spirit of the invention is to It allows for a certain degree of deviation from strict adherence to Such structural changes that “translate” into cleotides are and their analogues as long as the essential hybridization function occurs. I can do it.

Similarly, it is understood that species variation occurs between the various papillomavirus genera.

Various IFs! For example, E2, El, etc. are very similar for each species, but there are some differences. occurs. Changes in oligonucleotides and analogues that account for these mutations are , is specifically contemplated by the present invention.

Oligonucleotides and analogs used in the present invention can be synthesized using well-known techniques of solid phase synthesis. It can be manufactured conveniently and routinely. The equipment for such synthesis is The location is Applied Bio-systems. It is sold by several vendors such as s). However, like this Any other means of synthetic synthesis can also be used. Useful oligonucleotides can be conveniently obtained by fermentation methods, which are useful when large quantities of the substance are desired. It can be suitable in some cases. The actual synthesis of oligonucleotides is a routine procedure. It is sufficient that it is within the skill of the person in question.

Additionally, similar techniques can be used to synthesize other oligonucleotide analogs, e.g. It is known to produce ates and alkylated derivatives.

Testing the ability of antisense oligonucleotides to inhibit E2 expression The preferred assay to test is the well-documented transactivation of E2. based on the nature of the application. Svarzholz et al., J. Virol, 61.2128 ~2137 <1987>, reporter plasmid (E2RECAT) is E2 It was constructed to contain an E2-responsive element that acts as a dependent enhancer.

E2RECAT also contains the SV40 early promoter, early polyadenylation signal nal and chloramphenicol acetyltransferase genes (CAT) In the context of this plasmid, including Ru. The dependence of CAT expression on the presence of E2 makes this plasmid C127 cells transformed with 1, uninfected C127 cells and E2RF, CAT and transfected into 127M cells co-transfected with E2 expression vector. Tested by

Antisense oligonucleotides target the major E22 transactivator R It was designed to target NA (Figure 6). Targets include, but are not limited to, mR NA CAP region, translation initiation codon, translation termination codon and polyadenylation sig There was a naru. 15 or 30 residue oligonucleotides complementary to N targets. The internucleoside linkage of the wild-type phosphodiester or the modified phosphorothioester was synthesized using a straight internucleoside linkage. Targeting the mRNA CAP region oligonucleotide (11751) and E2 transactivator The translated codon (11753) was 1 micromolar in a preliminary screen. was found to reduce E2 transactivation (Figure 7), E2 Oligonucleotide 117 targeting the translation initiation codon of transrepressor 56 (Figure 6) shows that transrelation is demonstrated by and increases CAT activity. Similar lengths and salts were able to provide partial relief of pressure (Figure 7). base composition, but targeted other parts of E2 mRNA as well as other non-sensory controls. Other oligonucleotides were unable to confer antisense effect. General oligonucleotides with temporary mutations of phosphorothioate internucleoside linkages. Otide is an otide of the same sequence with naturally occurring phosphodiester internucleoside linkages. It was more effective than oligonucleotides. Presumably this is in the serum and cells increased resistance of phosphorothioates to nucleolytic enzymes contained within This is due to The dose-response curve shows the 50% inhibitory concentration (IC5o) of 11753. ranges from 50 to 100 nanomoles, but the IC5o of 11.751 is more than 10 times The transactivation of E2 is in the range of 500 nmoles (Figure 8). Successful antisense translation initiation codons of beta and trans-repressors Once identified as a target, an additional set of phosphorothioates can be added to that region with more attention. Designed to be deeply scrutinized (Figure 9), these data allow us to determine the appropriate AUG. The capped 15-20 mer oligonucleotide It has been shown that it can inhibit transrepression or transrepression ( 10 and 41).

The biology of BPV-1 confirms the consequences of E2 transactivator reduction in the field. To identify the antisense oligonucleotide, BPV-1 tested for the ability to inhibit or reduce transformation (Figure 12), 117 The dose-response curves for 51 and 11753 showed that the I Cci of 11753 .

is in the range of 10 nanomoles, but ICs o of 11751 is in the range of 100 nanomoles. (Figure 13), but these two compounds in this assay This 10-fold difference in ICso indicates that the translation initiation codon is a better target. similar to that observed in assays of transactivator inhibition suggesting that be.

Antisense targeting E2 against its ability to replicate BPV-1 into the genome To test the effect of oligonucleotides, stably transfected with BPV-1 The transfected l-38 cells were treated with I1751 and 11753, and the viral DNA A was quantified (Fig. 14), 48 hours after treatment with 1 micromolar IAI cells. The viral DNA copy number for each base was reduced by a factor of approximately 3. this During the assay, cells divided two to three times. This data indicates that the virus DNA 1, suggesting that it could not be replicated in parallel with the cell's DNA. do.

To test the effect of 11753 on E22 protein formation, l-3841 The cells were metabolically labeled and immunoprecipitated using an E2-specific monoclonal antibody.

Cells not exposed to oligonucleotide or sense or cells treated with inappropriate oligonucleotides contain the 46kd E2 protein. quality is present (Figure 15), treated with E2-targeted oligonucleotides. The 46kd band was missing in the cells that were translating the oligonucleotide. This suggests that it operates by blocking bridalization.

The oligonucleotides and oligonucleotide analogs of the present invention can be used in diagnosis, therapy, etc. , and can be used as research reagents and kits. For therapeutic use The oligonucleotides or oligonucleotide analogs can be added to papillomaviruses Infections, such as hand warts, foot warts, laryngeal warts, genital warts, and warts Epidermolysis dysplasia, flat neck warts, cervical epidermis neoplasia, or papillomatosis Administer to animals suffering from any other infection related to the genus. II, the present invention Preferably, the therapeutic agent is applied locally or intralesionally, transdermally or intralesionally. Other modes of administration may also be useful, such as intramuscularly. mixed into suppositories At present, there are 9 oligonucleotides of the present invention that are considered to be extremely useful. The use of leotide and oligonucleotide analogues in prophylaxis may also be useful. It will be done. For example, drugs can be used as coatings on condoms, etc. This can be achieved by providing Use of pharmacologically acceptable carriers Well, it's suitable for those who want to do it.

The present invention further provides oligonucleotides of the present invention that are useful in diagnosis and in research. Nucleotide and oligonucleotide analogs are highly effective against papillomavirus spp. Since hybridization occurs, the Sanderutsch method and other assays take advantage of this fact. It can be assembled easily. with oligonucleotides or analogs.

High contrast with Papillomavirus spp. present in a sample suspected of containing Papillomavirus spp. Equipment with means for detecting hybridization is routinely available. Wear. Such equipment may include enzyme conjugation, radiolabeling or any other suitable assay. One example is the output system. Detecting the presence or absence of papillomavirus spp. Also suitable may be kits for the purpose of

Example 1 Inhibition of BPV-IE2 expression by antisense oligonucleotides C127411 transformed with BPV-1! Place the cells into a 12-well plate.

24 hours before transfection with E2REI, antisense oligonucleotide By adding cleotide to the growth medium at final concentrations of 5.15 and 30 mmol. Pre-treat the cells. The next day, cells were phosphorylated with 10 μg of B2REICAT. Transfect by acid calcium precipitation. 10 μg of E2REICAT and 10 μg of carrier DNA (PUC19) in a final volume of 250 μl of H2O. , and incubate for 30 minutes at room temperature. Add 100 μl of this solution to each test well. Incubate at 37°C for 4 hours. After incubation, cells Give a glycerol shock for 1 min at room temperature using lycerol. shock After feeding, cells were washed twice with serum-poor DMEM and supplemented with 10% fetal bovine serum and original DMEM containing antisense oligonucleotide at a concentration of . Tran Harvest cells 48 hours after transfection and assay for CAT activity. .

To determine CAT activity, cells were washed twice with phosphorus MM* solution and scraped off. Collect. Cells were placed in 250 mmol Tris HC1, pH 8,0,100 μm. Resuspend and disrupt by freeze-thaw three times. 4ft cell extract 24 μl is used for each assay. For each assay, 25 μm of NaoOchi and M cell extracts were prepared as follows: .

4 mmol acetyl coenzyme A, 5 μl, H2O 18 μl and 14C-chlorampf. 1.5 ml Etzbendorf Mix together in a tube and incubate for 1 hour at 37°C. incube After lysis, chloramphenicol (acetylated and non-acetylated forms) is extracted with ethyl acetate and evaporated to dryness. Resuspend sample in ethyl acetate 25μm Let, spot on a thin layer chromatography plate, and chloroform: Thin layer chromatography, chromatography with methanol (19:1) is analyzed by autoradiography. Acetylated and non-acetylated The spot corresponding to 14C-chloramphenicol was placed on a thin layer chromatography plate. cells and counted by liquid scintillation for quantification of CAT activity. Ru. Antisense oligonucleotides that reduce CAT activity in a dose-dependent manner Considered positive.

Example 2 Inhibition of fPV E2 expression by antisense oligonucleotide Assays for inhibition of HPV E2 by antisense oligonucleotides It is essentially the same as that of PV-IE2. Appropriate website for HPV test Vs to CV-1 or A43 using the calcium phosphate method described above. 1 cell co-transfected with PSV2NEO cells. DNA The imported cells were selected by culturing them in a medium containing antibiotic 0418. The 0418It cells were then analyzed for HPV DNA and RNA. do. Cells expressing E2 are used as target cells for antisense studies. Each a After pretreatment of cells as described above for antisense oligonucleotides, E. 2 REICAT transfection and CAT transfection as described above. Performing assays for activity, antisense oligonucleotides show that they behave in a dose-dependent manner. It is considered to have a positive effect if it can reduce CAT activity. .

Example 3 Inhibition of HPV E7 expression by antisense oligonucleotide HP V-16 El transactivates the AdE2 promoter It turns out that it is possible to do this [Phelps.

W.C.), E.C.L., Munger-Gay (in) and H.C.B.M., 1988, transactivation similar to adenovirus EIA. Human papillomavirus type 16 E7 encoding tivation and transformation functions Gene (The Human Papi [Iomavirus Type 16 E7 Gene Encodes Transactivation andT transformation Functions 51m1lart.

Those of Adenovirus EIA), Ce1l, 5uni539 ~547], and to monitor this activity, we used the AdE2 promoter (AdE2C Construct a plasmid containing the chloramphenicol transferase gene under the control of AT). build Under this assay condition, CAT expression is dependent on HVP El expression. child For assays, the cell line expresses HPVE7 under the control of the SV40 early promoter. cause to contain; For each antisense oligonucleotide, pre-incubate cells. After pretreatment as described above, transfection with AdE2CAT was carried out. CAT activity is then analyzed as described above.

Example 4 Inhibition of BPV-I El expression by antisense oligonucleotides harm BPV-1 El has been shown to be a regulator of viral genome replication. Ta. To test the effect of antisense oligonucleotides on viral replication. To do this, C127 cells BPV-1 were infected with oligonucleotides at a final concentration of 5. E1-specific antibodies by adding them to the growth medium at 15 and 30 micromolar It is evaluated as such. In addition, antisense oligonucleotide viral genome copies The effect on -number is determined by Southern analysis on total genomic DNA.

Example 5 BPV-1 antisense antibody against experimentally induced bovine fibropapilloma treated with the same oligonucleotide as well as the control. induce regression of fibropapilloma M., 1985, Bovine papillomavirus (Bovin) containing multiple transforming genes. e papillomavirus contains-Roux 3-on-1,1 - Sulfurization was carried out after each coupling using carbon dioxide [I.R.B. - (Iyer, R.P.), Phillips, L.R. L, R,>, Egan W., Segan Jay (R egan, J.) and Beaucage, S.L. ), 1990.3H-1,2-pentudithiol-3-one-1,1-dioxy Automation of sulfur-containing oligodeoxyribonucleotides using sulfur-transferring reagents The Automated Synthesis of Sulfur -Containlng 011g.

deoxyribonucleotides Using 3H-1,2-Be nsodithiol-3-one 1.1-Dioxides as aSu lfur-Transfer Reagent), J, Or, Chem.

, 1:4693-4699], increasing to ensure complete thiolation. Oligonucleotides were extracted from the capped 0 synthetic matrix after each sulfurization step. After cleavage, deprotection and detriylaton, Oligonucleotides were ethanol precipitated twice from NaCl and suspended in water. .

Oligonucleotide concentration was determined by optical density at 260 nm. 41 For use in cell culture assays, oligonucleotides are routinely diluted to 10 0 micromol stock and stored at 80°C until use. oligonucle The purity, integrity and quantity of the otide preparation samples were determined by Maniatis. A 20% acrylamide 7 molar urea gel (4 Determined by electrophoresis on 0 cm x 20 cm x O, 75 cm Mani at i s, T, Fritsch E.F. Fritsch, E.F.) and Sambrook, J.

J, ), Mo1ecular C1onin: Laboratory Manual (ALab oratory Manual): Cold Spring Harbor Laboratory Lee (Cold Spring Hrbor Laboratory); New Electrophoresed oligonucleotides were prepared using Sigma (Sigma) l-Ethyl-2[3-(1-ethylnaptole[ 1,2-d]-thiazolin-2-ylidene)-2-methyl-10benyl[nagt dyed with ``Sting All'', which is a 1,2-d]-thiazolium bromide. visualized in gel by [Dahlberg A. erg, A, E,), Digman, C, W ) and Peacock.

A, C,), 1969, J, Mo1. Biol, 41:39.

Example 7 Molecular construct E2 chloramphenicol acetyltransferase (CAT) reporter used in this study - Plasmids were previously described [Svartsholz B.A., Vern J.D. I.C. and H.C.B.M., 1988, Bovine milk HIl virus type 1 Demonstration of cooperativity between E2 binding sites in E2 trans-regulation (Evid ence for Cooperation between E2 Bin ding 5ites in E2 trans-regulatfon of Bovine Papilomavirus T’yPe 1), J, Vlr oll, 62:3143-31501. Briefly, the 82 reactive elements of BPV-1 A certain E2RE1 (nt 7611-7806) was developed using oligonucleotides. Reconstituted psVZc with deletion of SV40 enhancer Sph, 1 fragment cloned into AT. Expression of CAT from this plasmid allows for full-length It was found that it depends on E2. Plasmid C59 carries the SV40 promoter and E2 cDNA expressed from the enhancer and described in detail elsewhere. [Yang K.C., Okayama H. and H.B.M., 1985, a bovine papilloma virus (Bovine p. apilomavirus contains multfple trans formlng genes), Proc.

Natl, Acad, Sci, USA, 82:1030-1034], two types of A full-length HPV-11 E2 expression construct was produced. IPV115 is pMsG (Cat. No. 27- HPV-11 (nt2665-4) cloned into the SmaI site of 988), and TPV118 contains Xmnl11 pieces of pSVL (77?Sia, The same HPV cloned into the Sma1 site of Catalog No. 27-4509) Contains ~11 Xm mouth ■ fragments.

Example 8 Cell line Mouse C127 cells [Dvoretzky, 1., Scoba - Schober, R. and Rollei D., 1980, Cattle Focus assay of papillomavirus type 1 in mouse cells (Focus Assay) in Mouse Cells for BovinePapHlomavir us type, 1), V soil ro soil earth engineering, L O3: 369-375, 10 % fetal bovine serum, penicillin <100 U/ml), streptomycin <110 Darbetsu Commodi supplemented with 0 u/ml) and L-glutamine (4 mmol) Grown in Fido Eagle medium. l-38 cell line is purified BPV- was derived from a single focus of C127m cells transformed by Cowsert, L, M, Lake, P. ) and Jenson, A.

B.), 1987, Bovine papillomavirus 1 defined by monoclonal antibodies. Structure outline and conformational epitopes (Topograph) ical and conformationalEpltopes of B ovine Paplloma virus type I Defined b y Monoclonal Antibodies), JNCI, 79:1053 -1057].

Example 9 Oligonucleotide inhibition of E2-dependent transactivation assay Labels that inhibit E2 transactivation or transrepression The calcium phosphate precipitation method as described by Graham et al. Therefore, transfection [Graham, F.

L, ) and van der Eb A.

J.), 1973, A new technique for the infectivity assay of human adenovirus 5 DNA ( A New Technique for the As5ay ofInfe ctivity of Human Adenovirus 5 DNA), V Jiro Engineering Σ, 52: 456-461 L sediment @ 20 μg in 1 mL is all DNA Met. In each 60 mm dish, prepare 200 pellets containing 4 micrograms of DNA. A microliter was obtained. 4 hours after adding the precipitated DNA, aspirate the supernatant. cells were treated with 15% glycerol and incubated with Frost, E. , ) and Williams, J., 1978 , temperature sensitivity and host range mapped by adenovirus type 5 with marker protection. Mutation (Mapping Temperature-3 intensive and host-range mutation of Adenovirus type 　5　by　Marker　Re5cue), Ekaji Nitachi 1dandanヱ, Construction: 39~ [50], after washing, refeeding the cells with medium containing the oligonucleotide at its original concentration; Incubated for 48 hours.

Although a number of specific embodiments have been set forth, the invention is limited only by the scope of the following claims. It is limited.

Sequence list (1) General information: (1) Applicant: Crooke.

5tan, 1ey T heat, Mirabell Christopher Kay <Mirabell i, Christopher K.), Elaker Debbie, Jay (Eck er, David J.), Coswert, L.M. ex　M,) (it) Title of the invention: Antisense oligonucleotide inhibitor of papillomavirus genus I I (iii) Number of arrays: 6 (1v) Address: (A) Address: Woodcock Washburn Carla Matskivy'〉Ann De Norris (Woodcock Washburn KurtzMackiew icz & Norris) (B) District: One Liberty Lewis (One L) ibertyPlace>46tll (C) Car name: Philadelphia (D) State name: Pennsylvania (E) Country name: United States (F) Postal code: 19103 (V) Computer readable format: (A) Medium size: small disk, 3.5 inches, storage 21.44Mb (B) Computer Computer: IBM PS/2(C) Operating system: PC-DOS (D>Software: Wordperfect 5.0 (vi) Recent application materials: (A) Application number: PCT/US90107067 (B) Application number: 1990 February 3rd (C) Classification: (vii) Prior application materials: (A) Application number: (B) Date of engagement: (wijt) Patent attorney/agent information: (A) Name: Jane MasseyLlcat a) (B) Registration number: 32,257 (C) Inquiry/case reference number, rsIs-0033(ix) communication information: ' (A) Speak: (215) 568-3100 (B) Fax: (21 5) Information on 568-3439 (2) Sequence identification number: 1: (i) Features of array (A) Length: 15 (B) Type: Nucleic acid GCTTCCATCT TCCTC15 (2-in sequence identification number: 2 information: (i) Features of array <A) Length: 16 (B) Type: Nucleic acid (xl) Sequence type: Sequence identification number: 2: GCTTCC: ATCT TCCTCG 16(2) Sequence identification number: 3 information: (1) Characteristics of array (A) Length = 17 (B) Species M: Nucleic acid (xi) Sequence type: Sequence identification number: 3: TGCTTCCATCTTCCTCG 17(2) Information on sequence identification number = 4: (i) Features of array (A) Length: 18 (B)! ! Category 1: Nucleic acid (xi) Sequence type: Sequence identification number = 4: TGCTTCCATCTTCCTCGT 18(2) Information on sequence identification number = 5: (i) Features of array (A) Length: 19 CBIN vote = Nucleic acid (xi) Sequence type: Sequence identification number = 5: TTGCTTCCAT CTTCCTC GT 19 (2) Sequence identification number = 6 information: (i) Features of array (A) Length: 20 (B) Type dimorphic acid (xi>Sequence M type: Sequence identification number: 6: TTGCTTCCAT CTTCCTC GTC20M? to r-co CI’l Or4 tn t, o to n? In00 m n M M f? OO’f? ? ? FIG. 12 0UTC Exit 1751 Tomoe 1753 -L/14 FIG. 15 r+u+ l l abstract There may be an antisense relationship with papillomavirus messenger RNA. Oligonucleotides and oligonucleotide analogs are provided. Like that oligonucleotides and oligonucleotide analogues for diagnostic as well as research purposes. and can be used for treatment. According to a preferred embodiment of the invention, papillomavirus oligonucleotides and oligonucleotides to which the indexer RNA hybridizes. Tide analogs are provided. This oligonucleotide or oligonucleotides Mimics can inhibit the function of RNA and are therefore useful for example in the treatment of infections caused by papillomavirus. It is useful for According to a preferred embodiment, parts of the papillomavirus are susceptible to antisense attack. It's a target. In this way, E2. El, ET or E6-7 Methsenji RNA5 Oligonucleotides that hybridize with are preferably provided.

Submission of translation of amendment (Article 184-8 of the Patent Law) June 4, 1992 Wataru Fukasawa, Commissioner of the Japan Patent Office, France 1. Display of patent application 2. Name of the invention Antisense oligonucleotide inhibition of papillomavirus genus Faraday Avenue -2280 Name: Isis Pharmaceutica Loss 8 Incorbo Seated Shin-Otemachi Bi Le 206 Ward 5. 8 days for submission of amendment The scope of the claims 1. E2, El, El Manaha E6-7 messengers derived from milk uI virus genus - Oligonucleos containing 8 to 50 bases that can hybridize with RNA or oligonucleotide analogues of the aforementioned Metsenger R,NA said oligonucleotide that inhibits the function of said oligonucleotide when hybridized thereto. oligonucleotide analogs.

2. The above-mentioned E2 metsenger RNA is an E2 receptor of papillomavirus RNA. 2. The oligonucleotide or oligonucleotide according to claim 1, comprising a portion of an activator. Ligonucleotide analogs.

3. Oligonucleotide containing a base sequence according to at least one of the sequences shown in Table 2 or oligonucleotide analogs.

4. A method for modulating the expression of a papillomavirus genus, the method comprising: Metsenger RNA derived from the genus is added to the oligonucleotide according to claim 1.2 or 3. A method as described above comprising contacting with an otide or oligonucleotide analog.

5. A method for modulating the effect of papillomavirus infection in animals, the method comprising: The oligonucleotide or oligonucleotides according to claim 1.2 or 3. The aforementioned method comprising contacting with an analog.

6. Detecting the presence or absence of papillomaviruses in samples suspected of containing them. A method for producing a sample and an oligo according to claim 1.2 or 3. contact with a nucleotide or oligonucleotide analog: and hybridize. 2. The method as described above, comprising detecting zesion.

7. Detecting the presence or absence of papillomaviruses in samples suspected of containing them. A kit for delivering the oligonucleotide according to claim 1.2 or 3. or analogs: and a means for detecting hybridization. The kit mentioned above.

[Iyer, R.P., Phillips L.A. Phi I 1ips, L, R, - Egan, W.), Regan, J., and Beaucage-Niss.E. Beaucage, S. L., 1990.3H-1,2-penduzithio Sulfur-containing oligos using 3-one-1,1-dioxide as a sulfur rearrangement reagent Automated synthesis of deoxyribonucleotides dithfol-3-one 1,1-Dioxides as aSulfu r-Transfer Reagent), J, Or, Chenn.

In each 60 mm dish, collect 200 microliters of pellet containing 4 micrograms of DNA. Tutle was obtained.

Go, kill, 659 too FtG, 12 0IJTc Mouth 1751 Tomoe 1753 -L・14 international search report mmal　A$1’　4*Mallow-Fu voice

Claims (51)

[Claims] 1. An enzyme capable of hybridizing with messenger RNA derived from the genus Papillomavirus. oligonucleotides or oligonucleotide analogs, wherein said message is said oligo that inhibits the function of the protein RNA when hybridized thereto. Nucleotide or oligonucleotide analogs. 2. The messenger RNA is at least Papillomavirus E2 RNA. The oligonucleotide or oligonucleotide according to claim 1, comprising a partial sequence. Chido analogues. 3. The messenger RNA is at least one of Papillomavirus ElRNA. The oligonucleotide or oligonucleotide according to claim 1, comprising a partial sequence. Chido analogues. 4. The messenger RNA is at least Papillomavirus E7 RNA. The oligonucleotide or oligonucleotide according to claim 1, comprising a partial sequence. Chido analogues. 5. The messenger RNA is a small amount of Papillomavirus E6-7 RNA. The oligonucleotide or oligonucleotide according to claim 1, which comprises a partial sequence of both Reotide analogs. 6. At least the E2 transactivator portion of papillomavirus RNA The oligonucleotide or oligonucleotide according to claim 1, which is complementary to the moiety Otide analogue. 7. The transactivator portion is 244 of bovine papillomavirus-1. Oligonucleoacids according to claim 6 corresponding to nucleotides from 3 to 3080. tide or oligonucleotide analogs. 8. complementary to at least a portion of the 5' untranslated region of Papillomavirus RNA. The oligonucleotide or oligonucleotide analogue according to claim 1. 9. The 5' untranslated portion is from 89 to 304 of bovine papillomavirus-1. The oligonucleotide or oligonucleotide according to claim 8, which corresponds to a nucleotide. Reotide analogs. 10. The oligonucleotide according to claim 1, comprising about 3 to about 50 base units. or oligonucleotide analogs. 11. The oligonucleotide according to claim 1, comprising about 8 to about 25 base units. or oligonucleotide analogs. 12. 2. The oligonucleotide of claim 1, comprising about 12 to about 20 salt reference points. or oligonucleotide analogs. 13. The translation of the messenger RNA into protein is carried out by hybridizing with it. The oligonucleotide or oligonucleotide according to claim 1, which inhibits the formation of Creotide analog. 14. Oligonucleotide containing a base sequence according to at least one of the sequences in Table 2 or oligonucleotide analogs. 15. A method of modulating the expression of a papillomavirus, said papillomavirus Messenger RNA derived from the genus Papillomavirus was converted into messenger RNA derived from the genus Papillomavirus. An oligonucleotide or oligonucleotide analog capable of hybridizing with A body and hybridizes with the above messenger RNA function. contact with said oligonucleotide or oligonucleotide analog to inhibit The above method comprising touching. 16. The messenger RNA is at least one of papillomavirus E2 RNA. 16. The method of claim 15, further comprising a partial sequence. 17. The messenger RNA is at least one of Papillomavirus ElRNA. 16. The method of claim 15, further comprising a partial sequence. 18. The messenger RNA is at least one of the papillomavirus genus E7 RNA. 16. The method of claim 15, further comprising a partial sequence. 19. The above messenger RNA is a small amount of papillomavirus E6-7 RNA. 16. The method of claim 15, comprising an arrangement of at least a portion of a spider. 20. The oligonucleotide or oligonucleotide analog described above may be used to treat papillary tumors. Complementary to at least a portion of the E2 transactivator portion of I. genus RNA 16. The method according to claim 15. 21. The transactivator portion is 24 of bovine papillomavirus-1. 21. The method of claim 20, corresponding to nucleotides 43 to 3080. 22. The oligonucleotide or oligonucleotide analog described above may be used to treat papillary tumors. Claim 15 which is complementary to at least a portion of the 5' untranslated region of I. virus RNA. The method described in. 23. The 5' untranslated portion is from 89 to 304 of bovine papillomavirus-1. 23. The method of claim 22, wherein the nucleotide corresponds to 24. The oligonucleotide or oligonucleotide analog has a base unit 16. The method of claim 15, comprising from about 3 to about 50. 25. The oligonucleotide or oligonucleotide analog has a base unit 16. The method of claim 15, comprising from about 8 to about 25. 26. The oligonucleotide or oligonucleotide analog has a base unit 16. The method of claim 15, comprising from about 12 to about 20. 27. Claim 15: inhibiting the translation of said messenger RNA into protein. The method described in. 28. 16. The method according to claim 15, which inhibits translocation of said messenger RNA to the cytoplasm. How to put it on. 29. A method of modulating the effects of a papillomavirus infection in an animal, the method comprising: , an oligonucleotide that can hybridize with messenger RNA from the papillomavirus genus. oligonucleotide or oligonucleotide analogue of said messenger said oligonucleotide that inhibits the function of RNA when hybridized thereto; The aforementioned method comprising contacting with leotide or an oligonucleotide analog. 30. The messenger RNA is at least one of papillomavirus E2 RNA. 30. The method of claim 29, further comprising a partial sequence. 31. The messenger RNA is at least one of Papillomavirus ElRNA. 30. The method of claim 29, further comprising a partial sequence. 32. The messenger RNA is at least one of the papillomavirus genus E7 RNA. 30. The method of claim 29, further comprising a partial sequence. 33. The above messenger RNA is a small amount of papillomavirus E6-7 RNA. 30. The method of claim 29, comprising at least a partial sequence. 34. The oligonucleotide or oligonucleotide analog described above may be used to treat papillary tumors. Complementary to at least a portion of the E2 transactivator portion of I. genus RNA 30. The method of claim 29. 35. The transactivator portion is 24 of bovine papillomavirus-1. 35. The method of claim 34, corresponding to nucleotides 43 to 3080. 36. The oligonucleotide or oligonucleotide analog described above may be used to treat papillary tumors. Claim 29 is complementary to at least a portion of the 5' untranslated region of I. virus RNA. The method described in. 37. The 5' untranslated portion is from 89 to 304 of bovine papillomavirus-1. 37. The method of claim 36, wherein the nucleotide corresponds to 38. The oligonucleotide or oligonucleotide analog has a base unit 30. The method of claim 29, comprising from about 3 to about 50. 39. The oligonucleotide or oligonucleotide analog has a base unit 30. The method of claim 29, comprising from about 8 to about 25. 40. The oligonucleotide or oligonucleotide analog has a base unit 30. The method of claim 29, comprising about 12 to about 20. 41. 29. The method inhibits the translation of said messenger RNA into protein. The method described in. 42. The above infections can cause warts on the hands, feet, laryngeal warts, condyloma acuminata, and genital warts. Claim 4: Epidermolytic dysplasia, flat neck wart, or head intraepithelial neoplasia. The method described in 2. 43. Testing for the presence or absence of Papillomaviruses in samples suspected of containing them It is a method for issuing Hybridize the sample with messenger RNA from the genus Papillomavirus. contact with an oligonucleotide or oligonucleotide analog capable of The aforementioned method comprising detecting hybridization. 44. The aforementioned messenger RNA is derived from papillomavirus genera E2, E1, E7 and and at least a portion of the sequence of at least one type of E6-7 RNAs. The method according to item 43. 45. Papillomas containing oligonucleotides or oligonucleotide analogs as described above. Compatible with at least a portion of the E2 transactivator portion of viral RNA 44. The method of claim 43, which is complementary. 46. The transactivator portion is 24 of bovine papillomavirus-1. 46. The method of claim 45, corresponding to nucleotides 43 to 3080. 47. The oligonucleotide or oligonucleotide analog described above may be used to treat papillary tumors. Claim 43 is complementary to at least a portion of the 5' untranslated region of I. virus RNA. The method described in. 48. The 5' untranslated portion is from 89 to 304 of bovine papillomavirus-1. 48. The method of claim 47, wherein the nucleotide corresponds to 49. The detection step includes detecting a labeled protein encoded by the messenger RNA. 44. The method of claim 43, comprising detecting the formation of texture. 50. The detection step includes detecting a labeled protein encoded by the messenger RNA. 44. The method of claim 43, comprising detecting the effect of a protein. 51. Testing for the presence or absence of Papillomaviruses in samples suspected of containing them It is a kit for distributing Oligos capable of hybridizing with messenger RNA from the papillomavirus genus Nucleotide or oligonucleotide analogs and hybridization A kit as described above comprising means for detection.